Identification of potentially active tyrosine kinases in lung cancer

在博德研究所的托德·戈卢布的合作下，我们采用了Luminex试验来定量测定酪氨酸激酶中的酪氨酸磷酸化。活跃的酪氨酸激酶通常也与总的酪氨酸磷酸化增加有关。我们的方法在多重分析中捕获了150多个酪氨酸激酶，并使用泛磷酸酪氨酸抗体(4G10)检测了所有这些激酶中的酪氨酸磷酸化水平。我们使用150例人肺腺癌肿瘤组织和相应的邻近正常肺组织来分析这些酪氨酸激酶的酪氨酸磷酸化。我们鉴定了大约30个特定的酪氨酸激酶，与邻近的正常肺组织相比，肿瘤组织中存在差异磷酸化。这种潜在活性的酪氨酸激酶的例子有NTRK1、EphA2、DDR。然后，我们与MSKCC的Marc Ladanyi博士合作完成了纳米串测试，以在多重测试中测量大多数酪氨酸激酶的转录表达。有趣的是，正如我们预测的那样，几种酪氨酸激酶的表达至少在转录水平上没有变化，但酪氨酸磷酸化有显著的变化，它通常是激活的替代。目前，我们正在与Paul Meltzer博士在NCI Intral计划中合作，对大约1300个与癌症相关的基因进行测序，包括Luminex测试中使用的酪氨酸激酶，以有针对性地识别特定的突变。

In collaboration with Todd Golub at the Broad Institute, we have adapted a luminex assay to quantitate tyrosine phosphorylation in tyrosine kinases. Active tyrosine kinases are also generally associated with increased overall tyrosine phosphorylation. Our assay captured more than 150 tyrosine kinases in a multiplex assay and detected the level of tyrosine phosphorylation in all these kinases using a pan-phosphotyrosine antibody (4G10). We used 150 human lung adenocarcinoma tumor tissue and corresponding adjacent normal lung tissue to profile tyrosine phosphorylation of these tyrosine kinases. We identified around 30 specific tyrosine kinases differentially phosphorylated in tumor tissue compared to adjacent normal lung. Examples of such potentially active tyrosine kinases are NTRK1, EPHA2, DDR. We then completed a nanostring assay in collaboration with Dr. Marc Ladanyi at MSKCC to measure transcript expression of majority of tyrosine kinases in a multiplex assay. Interestingly, as we had predicted, expression of several tyrosine kinases do not change at least at the level of trasnscription, however there is significant change in tyrosine phosphorylation, which is generally a surrogate of activation. Currently, in collaboration with Dr. Paul Meltzer in the NCI intramural program, we are sequencing around 1300 cancer-related genes, including the tyrosine kinases used in our luminex assay to identify specific mutations in a targeted manner.